Zebrafish offers a powerful model system in which to study hematopoietic development due to its external fertilization, transparent embryos and ease of genetic manipulation. These characteristics allow mutagenesis screens to be performed in zebrafish that are not currently feasible in other vertebrate systems. Over 30 hematopoietic mutants have already been isolated through two large- scale chemical (N-nitroso-N-ethylurea or ENU) mutagenesis screens. We are characterizing and mapping one of these recessive mutants, vlad tepes, which has few or no blood cells. Using RNA in situ hybridization with markers along the hematopoietic pathway, we have demonstrated that vlad tepes has a block in the erythroid pathway with preservation of the myeloid lineage. We have mapped the mutation to linkage group 11 using half-tetrad analysis, and have determined its approximate distance from the centromere to be 2.2-3.2 cM. While GATA-1 maps to this location and is a likely candidate gene, analysis by RT-PCR, sequencing, and Northern analysis have not revealed significant alterations. Further mapping now suggests that GATA-1 is not the involved gene based on recombinants found for the GATA-1 locus. Additional informative markers are being used to refine the map position of the mutation. We are now performing an ENU mutagenesis to screen for defects in the myeloid pathway with RNA in situ hybridization using myeloid-specific markers. We have generated over 450 F1 progeny that can be screened for recessive phenotypes through haploid screening. Future studies will focus on identifying zebrafish mutants that demonstrate loss of expression of one or more myeloid markers. We have also isolated a zebrafish cDNA clone for CBFB (named zCBFB), a gene that regulates hematopoiesis and is involved in human leukemias. In biochemical analyses, zCBFb binds human CBFa2 and enhances its DNA binding. Expression of zCBFB in normal zebrafish embryos is observed in the posterior intermediate cell mass (ICM, the location of hematopoietic progenitor cells), Rohon Beard cells (sensory neurons roughly similar to dorsal root ganglia), cranial nerves, retina, branchial arches, hind brain, and jaw. This expression pattern is very similar to that in the mouse embryos. Expression of zCBFB in hematopoietic mutants (bloodless) is decreased or absent in the ICM and Rohon Beard cells. We have also analyzed the expression of zSCL and zGATA-1 in the same blood mutants to ascertain the relative order of these transcription factors to zCBFB in zebrafish hematopoiesis. Most of our results indicate that the three transcription factors function in the same hierarchical order as in mice: SCL, CBFB, and GATA-1. The exception to this rule is the cloche mutant, in which zSCL expression is lost while zCBFB expression remains in the ICM region. This result suggests that CBFB and SCL may function in independent pathways. - genetics, hematology, bone marrow, leukemia, gene mapping (non-human), stem cell research